Hemodynamic molecular imaging of tumor-associated enzyme activity in the living brain

Abstract

Molecular imaging could have great utility for detecting, classifying, and guiding treatment of brain disorders, but existing probes offer limited capability for assessing relevant physiological parameters. Here, we describe a potent approach for noninvasive mapping of cancer-associated enzyme activity using a molecular sensor that acts on the vasculature, providing a diagnostic readout via local changes in hemodynamic image contrast. The sensor is targeted at the fibroblast activation protein (FAP), an extracellular dipeptidase and clinically relevant biomarker of brain tumor biology. Optimal FAP sensor variants were identified by screening a series of prototypes for responsiveness in a cell-based bioassay. The best variant was then applied for quantitative neuroimaging of FAP activity in rats, where it reveals nanomolar-scale FAP expression by xenografted cells. The activated probe also induces robust hemodynamic contrast in nonhuman primate brain. This work thus demonstrates a potentially translatable strategy for ultrasensitive functional imaging of molecular targets in neuromedicine.

Designing Protein‐Based Probes for Sensing Biological Analytes with Magnetic Resonance Imaging

“…Sensitive techniques to assay protease activity in vivo are therefore in critical demand both for basic research on disease pathways as well as to facilitate drug development efforts. While several synthetic MRI agents have been developed to sense protease activity, biomolecular ( i. e ., protein‐or peptide‐based) reporters are available for two proteases, cathepsin B [64] and fibroblast associated protein (FAP), [65] both of which are highly expressed in tumors and tumor‐associated stromal tissue. The cathepsin reporter was designed on the basis of poly‐L‐glutamate (PLG), which can be cleaved by protease action to release smaller glutamate‐containing peptides and individual glutamate moieties [64] .…”

“…While systemic injection of PLG allowed cathepsin activity to be monitored in rat brain tumors (Figure 4A, Table 1), the sensitivity and precision of this technique were limited, millimolar concentrations of the PLG probe generating ∼19 % CEST contrast with modest statistical fidelity ( p‐value in the 0.048 range). In contrast to PLG, detection of FAP [65] was accomplished with much greater sensitivity on the basis of probe‐induced vasodilation, similar to the mechanism adopted for calcium sensing with NOSTICs. Here, the probe comprises a calcitonin gene related peptide (CGRP), a 37‐residue molecule that binds to a heterodimeric receptor (known as CLR/RAMP) in vascular smooth muscle cells causing dilation of blood vessels, thereby producing MRI contrast [39] .…”

“…PKA ~8 % < 4 % -6.8 -protamine sulfate [63] kinase ~40 % ~25 % -6.9 -poly-L-glutamate [64] cathepsin ~22 % ~26 % 19 % -160 mg/kg [c] -CGRP [65] FAP --5 % 3.7 1 pmol [d] healthy cell morphology at implantation site (striatum) in histological sections of the rat brain small molecule drug without any effect on the BOLD response.…”

“…While systemic injection of PLG allowed cathepsin activity to be monitored in rat brain tumors (Figure 4A, Table 1), the sensitivity and precision of this technique were limited, millimolar concentrations of the PLG probe generating ~19 % CEST contrast with modest statistical fidelity (p-value in the 0.048 range). In contrast to PLG, detection of FAP [65] was accomplished with much greater sensitivity on the basis of probe-induced vasodilation, similar to the mechanism adopted for calcium sensing with NOSTICs.…”

Biomarker‐Responsive Nanosystems for Chronic Disease Theranostics

“…Pathological biomarkers are closely associated with the occurrence and progression of diseases such as cancer, CVDs, and neurological diseases. [ 14–18 ] Various features within pathological microenvironments include acidic pH, [ 19,20 ] hypoxia, [ 21–25 ] overexpressed reactive oxygen species (ROS), [ 26–36 ] glutathione (GSH), [ 37–43 ] enzymes, [ 44–48 ] and abnormal metal ions, [ 49–53 ] among others, which inspires the development of endogenous stimuli‐responsive nanomaterials [ 54–60 ] for the diagnosis and treatment of chronic diseases.…”